Multiplex Quantitative PCR Detection Of Major Pathogenic Bacteria In Chicken

Chicken has become the major consumer goods in people's daily life.In the process of production,processing and transportation,chicken is easily contaminated by various pathogenic bacteria,which brings safety risks to chicken and also causes food poisoning easily.Therefore,it is of great significance for chicken safety and food poisoning events to understand the contamination of pathogens in chicken and establish a method for rapid detection of food-borne pathogens.The method can ensure the safety of chicken,and has important significance to the chicken industry.1.In order to establish a rapid detection method for salmonella in chicken,primers were designed according to the invA gene of salmonella invasive protein,and SYBR Green I quantitative PCR method was established and tested on chicken samples.The results showed that the quantitative PCR method had no crossover with the control Staphylococcus aureus,Proteus mirabilis,Enterococcus,Streptococcus,Pseudomonas aeruginosa,Bacillus subtilis,Enterobacter cloacae,and the minimum detection concentration was 6.47x10~1 CFU/mL The repeat coefficient of variation for mL was0.3%.The detection rate of Salmonella in chicken meat was 88.1%.These point out this experiment establish a quantitative PCR method that is specific,sensitive,and stable,and can be used for rapid detection of Salmonella in chicken.2.In order to establish a rapid detection method for Staphylococcus aureus in chicken,primers were designed according to the thermostable nuclease nuc gene of Staphylococcus aureus,and the quantitative PCR method of SYBR Green I was established and the chicken samples were tested.The results showed that the quantitative PCR method had no crossover with the control Salmonella,Proteus mirabilis,Enterococcus,Streptococcus,Pseudomonas aeruginosa,Bacillus subtilis,Enterobacter cloacae,and the minimum detection concentration was 2.05x10~1 CFU/mL.The coefficient of variation is between 0.6%and 1.3%.The detection rate of Staphylococcus aureus in chicken was 92.1%.These point out this experiment establish a quantitative PCR method that is specific,sensitive,and stable,and can be used for rapid detection of Staphylococcus aureus in chicken.3.In order to establish a rapid detection method for Proteus mirabilis in chicken,the primers were designed according to the qrrA gene of Proteus mirabilis urease,and the quantitative PCR method of SYBR Green I was established and the chicken samples were tested.The results showed that the quantitative PCR method had no crossover with Salmonella,Staphylococcus aureus,Enterococcus,Streptococcus,Pseudomonas aeruginosa,Bacillus subtilis,Enterobacter cloacae,and the minimum detection concentration was 5.04 x10~1 CFU/mL The coefficient of variation is between0.9%and 1.3%.The detection rate of Proteus mirabilis in chicken was 72.3%.These point out this experiment establish a quantitative PCR method that is specific,sensitive,and stable,and can be used for rapid detection of Proteus mirabilis in chicken.4.In order to establish a rapid detection method of Enterococcus faecalis in chicken,primers were designed according to the sodococcus superoxide dismutase sodA gene,and a single-plex SYBR Green I quantitative PCR method was constructed and the chicken samples were tested.The results showed that the quantitative PCR method had no crossover with Salmonella,Staphylococcus aureus,Proteus mirabilis,Streptococcus,Pseudomonas aeruginosa,Bacillus subtilis,Enterobacter cloacae,and the minimum detection concentration was 3.05x10~1 CFU/mL.The repeat coefficient of variation is between 1.0%and 1.3%.The detection rate of enterococci in chicken was11.9%.These point out there is specific,sensitive and stable in the establishment of quantitative PCR method,which can be used for the rapid detection of Proteus mirabilis in chicken.5.In order to establish a rapid detection method for pathogenic bacteria such as Salmonella,Staphylococcus aureus,Proteus mirabilis and Enterococcus in chicken,four pathogen-specific primers were designed,and muLtiple SYBR Green I quantitative PCR methods were established to detect chicken meat sold in Hebei.As a result,a multiplex quantitative PCR method was established,which did not cross the bacteria against the control Streptococcus,Pseudomonas aeruginosa,Bacillus subtilis,Enterobacter cloacae,the minimum detection concentration was 10CFU/mL,and the coefficient of variation of the repeated test was 0~1.3%.The detection rates of Salmonella,Staphylococcus aureus,Proteus mirabilis,and Enterococcus in chicken were 88.1%,92.1%,72.3%,and 11.9%,respectively.The multiplex PCR method established in this study is sensitive,specific and stable,and can be used for rapid detection of Salmonella,Staphylococcus aureus,Proteus mirabilis and Enterococcus in chicken.The multiple quantitative detection method established in this paper can simultaneously detect Salmonella,Staphylococcus aureus,Proteus mirabilis and Enterococcus in chicken.The method can evaluate the pollution degree of chicken and avoid the risk early,which is of great significance to the quality and safety of chicken.